A major problem in developmental biology concerns the specification of identities along linear axes. Axial development frequently involves serial structures, as for example somites, which develop differentially depending on their position. Seemingly diverse developmental processes such as the formation of the cranial nerves, vertebrae, limbs, and the urogenital system all share the feature of axial specification that may involve common underlying developmental mechanisms. There is growing support for this integrative concept based primarily on the discovery of the homeobox genes which are highly conserved throughout multicellular organisms and involved in axial specification. The Principal Investigator will examine the molecular basis of homeobox gene activation associated with the specification of developmental identity. Hoxc-8 was chosen because it is representative of the medial class of Hox genes and because a wealth of information exists for this gene. Three major approaches to the analysis of Hoxc-8, all of which involve different aspects of its transcriptional control, will be taken. The first will identify enhancer motifs using reporter constructs in transgenic mice. Experience has shown this approach to be reliable, efficient, and rapid. The transgenic system will be combined with the use of YACs containing the mouse Hoxc cluster, and a novel clasper vector that permits the precise dissection of YACs by recombination. These new tools facilitate the detection of enhancer elements operating at substantial distances from the promoter, enhancer sharing between Hox genes, and genetic complementation. The second will characterize transcription factors regulating the expression of Hox genes by interaction with the defined enhancer motifs. Attention will be given to the combinational interaction of these proteins, a possibility underlined by their preliminary findings. The third will investigate functional aspects of Hoxc-8 enhancers at the molecular level using in vivo ligation-mediated PCR footprinting, using embryonic tissues, transgenic mice, and transformed cell populations.